A critical element of Pseudomonas aeruginosa pathogenesis is the ability to forms biofilms in the lungs of cystic fibrosis (CF) patients and on many other surfaces including wounds. Bacteria within biofilms produce one or more exopolysaccharides (EPS) that act as a scaffold, holding biofilm cells together and to a surface. In the prior funding period, we discovered that P. aeruginosa expresses an EPS designated Psl, which plays a critical role in cell surface interactions and biofilm formation. Our overall objective is to determine the role of Psl in biofilm development, structure, and P. aeruginosa pathogenesis. Aim 1 will focus on defining the mechanism of biofilm matrix formation mediated by the Psl pentasaccharide. In the second aim, the regulation of Psl expression will be investigated. The hypothesis that both transcriptional and post-transcriptional regulatory mechanisms are involved in controlling expression of the psl operon will be tested. Finally, experiments in aim 3 will test the hypothesis that Psl contributes to P. aeruginosa pathogenesis by modulating interactions with host immune cells. The work completed in the prior funding cycle as well as that the tools and models developed thus far sets the stage for answering key questions regarding biofilm matrix composition, the regulation of genes responsible for producing this matrix, and the role of Psl in modulating interactions of P. aeruginosa with the host. Since the matrix provides a critical protective role as well as a scaffold for the developing biofilm, agents aimed at disrupting the matrix would have therapeutic value. A thorough analysis of Psl will lead to the development of such agents and improve the quality of life for CF patients as well as individuals with other P. aeruginosa infections that involve biofilms.